Ionizing radiation (IR) has severely damaging effects on biological material. In the water based environment of the cell, IR triggers formation of free radicals that attack macromolecules. DNA repair pathways and a variety of antioxidant defense systems are thought to be central to radiation resistance. However, the role of RNA repair to this resistance is unknown. Stable and unstable RNA (tRNA/rRNA and mRNA, respectively) are damaged under a variety of stress conditions but are often presumed to be degraded after these insults. However, RNA is repaired under select circumstances including mRNA ligation in the unfolded protein response and sealing of breaks in the anticodon stem loop of tRNA . We hypothesize that additional examples of RNA repair are awaiting the appropriate lens. In this research-training plan, I propose to investigate the mechanism of RNA repair after IR utilizing the extremely radiation resistant cell Deinococcus radiodurans as a model system. Specifically, I will focus on the biochemical, structural, and physiological investigation of a radiation induced RNA repair enzyme, D. radiodurans RNA ligase (DraRnl). DraRnl is an ATP-dependent, dsRNA specific ligase that seals 5-PO4 and 3-OH nicks in a template dependent fashion. DraRnl is highly expressed in the early recovery phase (0-5 hr) after high gamma radiation doses (>10 kGy), suggesting an important physiological role for RNA ligation during radiation recovery. Aim 1 is to biochemically characterize DraRnls mechanism of action utilizing a variety of damaged substrates including those with mismatches, abasic sites, and oxidized bases at the break site. Preliminary data reported here demonstrates that DraRnl has a range of fidelity dictated by specific structural distortions in the damaged substrate. Aim 2 is to solve the X-ray structure of DraRnl family member at various mechanical stages of RNA repair. To this end, we have cloned, purified, and initiated crystallization trials of WT and mutant DraRnl family members. Finally, D. radiodurans offers a useful model for in vivo analysis of radiation induced RNA damage and repair. Aim 3 is to investigate RNA repair in vivo using WT and mutant strains of D. radiodurans. Next generation RNA sequencing along with classical methods such as northern blots and pulse-chase analysis will be used to study these phenomena. I anticipate these studies will provide a rewarding research training experience, and also important new insights into how cells respond to radiation damage.